The work proposed here aims to answer several key questions pertaining to tumorigenesis: (1) How does the interaction between normal cells and tumor cells regulate tumorigenesis, (2) Does loss of cell polarity contribute to tumor development and progression and (3) Determine if tissue polarity can suppress tumor progression. These studies will provide insight into the role(s) of cellular and tissue polarity in tumorigenesis in a context that is physiologically similar to what occurs in vivo. Results from our proposed studies may open a new avenue for targeted therapy that focuses on restoring polarity in order to prevent or limit tumor growth and metastasis. (1) Does oncogene activation induce non-cell-autonomous effects in neighboring cells, and do normal cells impact the phenotype of transformed cells? I will transduce mammary stem cells with lentivirus expressing a RFP-Ras fusion and mix them at a low concentration with stem cells expressing GFP-Histone H2A, and transplanted into a host mouse, to regenerate chimeric mammary glands. I will ask if normal cells neighboring the transformed cells show increased proliferation or signaling as compared to cells not in contact with transformed cells. In addition, chimeric organoids will be grown in 3D culture, to track cell behaviors in real-time by 4D confocal microscopy. (2) Can polarity proteins cooperate with known oncogenes to promote tumorigenesis? Loss of polarity proteins is predicted to increase invasiveness of tumors, by increasing the ability of transformed cells to escape the epithelial sheets in which they arise. Using the model systems from Aim 1, I will coordinately express oncogenic Ras in conjuction with the depletion of Par3 or Par6 by shRNA. I will address whether the loss of polarity proteins might lead to an increased probability of metastasis by reducing intercellular adhesion and/or through a loss of apical/basal polarity. (3) Does overall tissue polarity act as a tumor suppressor? Breakdown of the myoepithelial layer is a prerequisite for tumor metastasis, which has led to a common belief that the myoepithelium acts as a tumor suppressor. I will address this hypothesis by selectively transforming the luminal epithelial cells in the presence or absence of a normal myoepithelial layer in both 3D organotypic cultures and in murine mammary transplants. I will ask if escape of mutant luminal epithelial cells in organoids and metastases in mice are enhanced by conditional ablation of the myoepithelium.